A multi-part research program has been designed to yield information about the expression of the transfer RNA in E. coli, the biosynthesis and role of modified nucleotides in E. coli RNA, and the organization and expression of the genes which code for the metabolically stable E. coli 4.5S and 6S RNAs. The biological activities of 16 undermodified forms of phenylalanine tRNA will be compared with the fully modified species in an effort to reveal the role of tRNA base modification in the cell. To elucidate the mechanism of dihydrouridine biosynthesis in tRNA, a dihydrouridylate synthetase will be enriched and characterized using as substrate, dihydrouridine-eficient tRNA from amino acid-starved relaxed control E. coli. In an effort to identify tRNA species whose synthesis and rate of turnover are regulated differently from that of bulk tRNA, a two-dimensional acrylamide gel electrophoresis procedure will be used to determine the levels of individual tRNA species in cells in a variety of metabolic states. The fine structure and transcriptional properties of the five-cistron operon for the major E. coli leucine tRNA will be determined from in vitro transcription experiments in which cloned DNA corresponding to this particular operon is used as template material. The chromosomal distribution of repeated DNA sequences complementary to 4.5S and 6S RNAs will be determined by analyzing RNA:DNA hybrids constructed from DNA fragments produced with sppcific restriction endonucleases.